Site-directed and deletion mutagenesis analyses were used to define the function of three shared human immunodeficiency virus (HIV)-1, HIV-2 and simian immunodeficiency virus (SIV) Nef structural domains: a) leucine- repeat motif (dimerization domain); b) autophosphorylation (kinase domain); and c) acidic `-helical (transcriptional activation or protein- protein interaction domain). Mutational analysis on heptad leucine positions 1 (residue 108), 2 (residue 115), and 4 (residue 129), respectively, within the leucine zipper-like sequence of the HIV-2 NIHZ Nef protein confirms that this region forms the dimerization domain of Nef. While leucine to serine substitutions at positions 1 and 2, and leucine to glutamine at position 4 did not abrogate ability of the protein to form stable homo-oligomers in vitro, a similar mutation in which heptad leucines at positions 2 and 4 were changed to proline residues, totally abrogated homo-oligomer forming ability. Similarly, mutational analysis of individual cysteine residues at positions 28, 32, and 55 affected the stability of the NIHZ Nef protein following reduction of disulfide bonds by dithiothreitol(DTT), and reduced the ability to form stable homo-oligomers. In addition, using deletion analysis and Nef-affinity chromatography with glutathione-S-transferase (GST) Nef- fusion proteins immobilized on glutathione agarose, we have identified several cellular factors that bind to the carboxyl termini (potential activation or protein-protein interaction domain) of the HIV-1 and HIV-2 Nef proteins. Among these factors are 4 or 5 cellular phosphoproteins expressed in Jurkat, CEM, HL60, and U937 human leukemic cell lines. A 16-17K highly phosphorylated serine/threonine phosphoprotein binds in a region of the HIV-1 Nef carboxyl terminus containing the sequence motif, His-Pro-Glu-Tyr/Phe-Tyr/Phe, that is absent in the HIV-2 Nef protein. A 35-36K and 60-62K phosphoprotein binds to a region containing the sequence motif, Leu-X-Trp-Lys/Arg-Phe-Asp-X-X-Leu, that is shared between the HIV-1 and HIV-2/SIV Nef proteins, as well as with the activation domains of human herpes simplex virus (HSV) VP16 and Epstein-Barr virus (EBV) Rta transcription factors. The p35-36 protein is a tyrosine phosphorylated protein that also binds to the SH-2 domain of PLC-gamma in immunoblots.
